Puromycin treatment leads to increased levels of polyubiquitinated proteins.

<p>293-K^b cells were treated for 4 hours with media alone, 20 µM MG132, or the indicated concentrations of cycloheximide and puromycin (µM). Lysates were subjected to Western blotting with FK2 (<i>upper panel</i>), a monoclonal antibody specific for mono- and polyubiquitinated protein conjugates. Beta-tubulin was probed as a loading control (<i>lower panel</i>). Results are representative of three independent experiments.</p

Treatment with puromycin increases the fraction of rapidly degraded polypeptides.

<p><i>A.</i> 293-K^b cells were labeled with [³⁵S]-Met for 10 minutes in the presence of 0 to 40 µM MG132. [³⁵S] incorporation was measured as in Fig. 1A and normalized to controls without proteasome inhibitor (<i>n</i> = 3; mean ± s.e.m.) <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met +/−20 µM puro and +/−20 µM MG132 for 5 minutes, then chased from 0 to 50 minutes in the presence of excess cold methionine, CHX and +/−20 µM MG132. DMSO is a solvent control for MG132. The chase was terminated at the indicated time points by the addition of TCA to cell suspensions to precipitate polypeptides. TCA precipitates were solubilized and [³⁵S] was measured by liquid scintillation counting (<i>n</i> ≥4; mean ± s.e.m.) <i>C</i> and <i>D</i>. Solubilized TCA precipitates from cells radiolabeled in the absence (<i>C</i>) or presence (<i>D</i>) of 20 µM puro were separated by tricine SDS-PAGE on 10% gels. Gels were dried and exposed to a PhosphorImager plate overnight. Note that for <i>D</i>, the darkness of the image has been enhanced in order to see the contrast in degradation rates between <i>C</i> and <i>D</i> more clearly.</p

MHC class I-peptide complex recovery assay using a fluorescent reporter encoding antigenic peptides.

<p><i>A.</i> Schematic of the modified NP-SIINFEKL-eGFP reporter (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Princiotta1" target="_blank">[2]</a>) containing eight additional tandem repeats of SIINFEKL (nine total) and its five flanking amino acids from the native ovalbumin sequence, NP-[SIINFEKL]₉-eGFP (Tandem Repeat x9 or TRx9). <i>B.</i> Validation of MHC I peptide stripping and recovery in TRx9-expressing cells. Biexponential scatter plots show single cell profiles of the mean fluorescence intensity (MFI) for eGFP on the <i>x</i>-axis and 25-D1.16 on the <i>y</i>-axis. Plots show fluorescence profiles immediately pre- (<i>left</i>) and post- (<i>middle</i>) peptide stripping, and after a 4 hour recovery (<i>right</i>).</p

Rapid degradation of premature translational termination products.

<p><i>A.</i> Denaturing immunoprecipitation of peptidyl-puromycins. 293-K^b cells were radiolabeled for 30 minutes with [³⁵S]-Met, 20 µM puromycin, and 20 µM MG132. Polypeptides were precipitated with TCA, solubilized, then subjected to a denaturing immunoprecipitation using either non-specific rabbit serum (negative IP control) or anti-puromycin serum. Results are representative of three independent experiments. <i>B.</i> 293-K^b cells were pulse labeled with [³⁵S]-Met and 20 µM puro and chased as described in Fig. 3B. Solubilized TCA precipitates were subjected to denaturing immunoprecipitation using anti-puromycin serum. [³⁵S] in the anti-puromycin immunoprecipitates was measured by liquid scintillation counting (<i>n</i> = 4; mean ± s.e.m.).</p

Puromycin stimulates the production of truncated polypeptides in a dose-dependent manner.

<p><i>A.</i> 293-K^b cells were radiolabeled with [³⁵S]-Met for 10 minutes in the presence of a linear range of puromycin concentrations from 0 to 20 µM. Radiolabeled polypeptides were visualized as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone-0051968-g001" target="_blank">Fig. 1B</a>. In the later panels, we analyze the [³⁵S] signals from the four regions indicated to the right of the gel. <i>B</i>. PhosphorImager signal intensities (arbitrary units) from selected lanes in <i>A</i>. The left side of the graph corresponds to the top of the gel while the right side of the graph corresponds to the bottom of the gel at the dye front. The highlighted regions correspond to the parts of the gel indicated in <i>A</i>. <i>C</i>. The effects of puromycin concentration on [³⁵S] signal for each of the highlighted gel regions in <i>A</i> and <i>B</i>. Results are representative of three independent experiments.</p

Time-dependent inhibition of MHC class I pathway function following puromycin treatment.

<p><i>A</i> and <i>B.</i> 293-K^b cells were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface K^b was conducted in the presence of varying concentrations of puro for one (<i>A</i>) and four (<i>B</i>) hours. During the final 30 minutes of the recovery, cells were treated with either distilled water ((−) peptide) or 5 µM SIINFEKL peptide ((+) peptide) to promote the export of K^b to the cell surface <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Day1" target="_blank">[31]</a>. Flow cytometry was used to measure total cell surface K^b and the MFI was normalized to untreated cells in the absence of exogenous SIINFEKL peptide (<i>n</i> = 3; mean ± s.e.m.; * <i>p</i><0.05 for (−) peptide vs. (+) peptide).</p

Effects of puromycin on the recovery of cell surface MHC class I-peptide complexes.

<p><i>A</i>–<i>E</i>. 293-K^b cells expressing the TRx9 reporter were stripped of cell surface MHC I peptides as in Fig. 5. Recovery of cell surface MHC class I-peptide complexes was conducted in the presence of varying concentrations of puro from 0 to 180 minutes. Flow cytometry was used to measure reporter eGFP fluorescence (<i>A</i>), as well as cell surface K^b-SIINFEKL complexes (<i>B</i>) and total cell surface K^b (<i>D</i>). To quantitate differences in the kinetics of MHC class I-peptide complex recovery, we normalized the MFI values of puro-treated cells to untreated cells for K^b-SIINFEKL (<i>C</i>) and total K^b (<i>E</i>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051968#pone.0051968-Qian1" target="_blank">[4]</a> (<i>n</i> = 5; mean ± s.e.m.) For <i>C</i> and <i>E</i>, * <i>p</i><0.05 compared to untreated samples. <i>F.</i> MHC class I-peptide complex recovery after 1 hour in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.) <i>G</i>. MHC class I-peptide complex recovery after 4 hours in the presence of varying concentrations of puromycin. MFI values are normalized to untreated cells (<i>n</i> = 3; mean ± s.e.m.).</p

Relative metabolomic outputs of U87MG cells subjected to UPR stress compared to unstressed cells.

<p>U87 cells were grown in Knockout DMEM medium with serum replacement as described above. Cells were harvested, washed, and replated in the same (fresh) medium with or without 1 mM DTT, and with 5 mM ¹³C-glucose, for 4 hrs prior to cell and media harvest and PCA extraction as described in Materials and Methods. ¹H-, ³¹P-, and ¹³C-NMR spectra were obtained and quantified; data analyses were conducted as described. Graphs compare metabolite components from untreated cells (set to 100%) vs treated cells; error bars show standard deviation, and * = p< 0.05 derived from Student’s <i>t</i> test comparing treated to untreated in averages of 3 separate experiments. (<b>A</b>) displays data for soluble metabolites. Cho = choline; Cr = creatine GSH = glutathione; Lac = lactate. (<b>B</b>) displays data for high energy phosphates and [1-¹³C] glucose uptake. P-Choline = phosphocholine; GP-Choline = glycerophosphocholine; PME = phosphomonoesters; PDE = phosphodiesters; PC = phosphocholine; GPC = glycerophosphocholine; P-Creatine = phosphocreatine; UDPG = uridine diphosphoglucose; Glc = glucose; Lac = lactate. (<b>C</b>) shows data for lipid compounds. MUFA = monounsaturated fatty acids; TAG = triacylglycerols; Glycerol-Plipids = glycerol phospholipids; PtdCholine = phosphatidylcholine; PtdEthanolamine = phosphatidylethanolamine; PUFA = polyunsaturated fatty acids; FA = fatty acids.</p

Identification of UPR signaling response patterns in high-grade glioma xenografts and cell lines.

<p>Human glioma xenografts grown in <i>nu/nu</i> mice were derived from U87MG, and U87+EGFR (wild type) (cell lines described in the text and Materials and Methods). (<b>A</b>) Northern blots of 10 µg total RNA from replicate tumors (n=3) and normal brain from <i>nu/nu</i> mice; 10 µg total RNA from U87 tissue culture cells (“cells”) treated with the reducing agent DTT ([+]) lanes) to induce the UPR. Note transcriptional upregulation of UPR-induced mRNAs for ER chaperones (GRP94, BiP/GRP78) and UPR signaling components (XBP-1, CHOP, ATF4, ATF6). Quantification of BiP/GRP78 (<b>B</b>) and GRP94 (<b>C</b>) mRNA expression compared to mean level of expression in normal murine brain (dotted line). (<b>D</b>) U87MG, U87+EGFR, and U87+EGFRvIII (U87 cells transfected with the tumor-specific EGFR mutant variant III [in-frame deletion of exons 2-7]) cells show greater UPR inducibility with 1 mM DTT (determined by Northern blotting for XBP-1 and CHOP messages) than do HeLa cells. (<b>E</b>) Human glioma xenografts were derived from U87MG, U87+EGFR, and from D245MG, from a patient-derived Duke high grade glioma (from the Duke Brain Tumor BioRepository). Immunoblot of replicate tumors (n=3) from xenograft glioma models and normal brain from <i>nu/nu</i> mice. Note upregulation of ER chaperones in tumor lysates vs brain lysates: GRP170/ORP150, GRP94, calnexin (CNX), ERp72, protein disulfide isomerase (PDI), calreticulin (CRT), homocysteine-induced ER protein (HERP), and ER membrane markers (Sec61α and translocon associated protein, TRAPα) relative to loading control (β-actin). GRP78/BiP protein expression was variable in our Western blot assays. Blots probing for actin as loading controls are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone.0073267.s001" target="_blank">Supplemental Figure S1</a>. Blots for GRPs 170 and 78, for ERp72 and TRAPα were replicate blots. Blots for GRP94, CNX, CRT, HERP, and Sec61α were stripped and reprobed for actin.</p

Primary tissue culture cells from newly-resected gliomas also display inducible elements of the UPR.

<p>Dissociated cell cultured from freshly-resected GBMs were grown under serum-free conditions and were treated (or not, “Cont”) with 1 mM DTT (“+DTT”) for 4 hrs. Cell cultures were harvested, and cells lysed described. Proteins were separated on SDS-PAGE and Western blotted and probed with the antibodies listed. Upregulation of some of the UPR components is evident. Actin probe is used as a loading control, from the stripped CRT blot. Other actin blots to verify loading are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone.0073267.s003" target="_blank">Supplemental Figure S3</a>.</p